Differences in accumulation of blunt- and cohesive-ended double-strand breaks generated by restriction endonucleases in electroporated CHO cells.

Restriction endonucleases (RE) have been used in cytogenetic studies to mimic the DNA double-strand break (dsb)-inducing action of radiation. In the experiments presented here, we have treated electroporated CHO cells with RE and have measured the resulting dsb using the filter elution technique under non-denaturing conditions (pH 9.6). PvuII, which generates blunt-ended dsb, gave rise to a significant number of measurable dsb. The frequency of the dsb induced by PvuII is shown to increase over a 3-12-h post-treatment incubation period, which implies that the RE is active in the cell for a considerable length of time. We postulate that the accumulation of dsb reflects a competition between enzymatic incision and repair of the DNA. The presence of araA, a known inhibitor of DNA synthesis, did not affect the frequency of PvuII-induced breaks indicating a lack of an inhibitory effect of araA on the repair of RE-induced dsb. Two RE which cause cohesive-ended dsb, namely BamHI and EcoRI, were found to be ineffective in giving rise to measurable dsb. Our interpretation of this is that for cohesive-ended dsb (caused by BamHI and EcoRI) the rate at which these breaks are rejoined matches or exceeds the rate of enzymatic incision and hence no dsb were observed. In the case of PvuII, the possibly slower rate of repair of blunt-ended termini would on this hypothesis result in the observed net accumulation of dsb.     

Induction of chromosomal aberrations by restriction endonucleases encapsulated in liposomes

We used liposomes to deliver the restriction endonucleases BamHI and SmaI into human heteroploid HEp-2 cells. With this method very low concentrations of enzymes (2 units/ml) were active in the production of chromosomal aberrations. SmaI, which produces blunt-ended double-strand breaks in the DNA molecule, induces chromosomal aberrations more effectively than BamHI, which produces cohesive ends. Our results indicate that liposomes are suitable vectors for introducing restriction endonucleases into cultured human cells.     

The induction of chromosome aberrations by restriction endonucleases that produce blunt-end or cohesive-end double-strand breaks

Restriction endonucleases have been used to study the involvement of specific types of DNA damages in the production of chromosome aberrations. In this study restriction endonucleases were introduced into viable CHO cells using osmolytic shock of pinocytic vesicles. We compared two cohesive-end cutters, Msp I (CCGG-2-base overlap) and Sau3A I (GATC-4-base overlap) with two blunt-end cutters, Alu I (AGCT) and Rsa I (GTAC). All 4 enzymes were effective at inducing aberrations. The 4-base overlap cohesive-end cutter Sau3A I was approximately as effective as the blunt-end cutter Alu I. We present evidence that cutting frequency rather than cut end-structure is important in determining efficiency of aberration induction. There is over-dispersion of the distribution of dicentrics and rings among cells, and the data could be fitted to a Neyman Type A distribution, a modified Poisson, that indicates that there is a probability distribution both for the entry of the enzyme into a cell nucleus and for the induction of aberrations once the enzyme has entered a cell nucleus. In addition, we used Alu I to determine the sensitivity of cells to aberration induction in the different stages of the cell cycle. Alu I induced aberrations in all stages of the cycle, chromatid-type in S/G2 and chromosome-type in G1. In agreement with data of others, there were variations in sensitivity with cycle stage, and changes in the proportions of the different aberration classes for chromatid-type aberrations.     

Recombinational repair of chromosomal DNA double-strand breaks generated by a restriction endonuclease

DNA double-strand break repair can be accomplished by homologous recombination when a sister chromatid or a homologous chromosome is available. However, the study of sister chromatid double-strand break repair in prokaryotes is complicated by the difficulty in targeting a break to only one copy of two essentially identical DNA sequences. We have developed a system using the Escherichia coli chromosome and the restriction enzyme EcoKI, in which double-strand breaks can be introduced into only one sister chromatid. We have shown that the components of the RecBCD and RecFOR 'pathways' are required for the recombinational repair of these breaks. Furthermore, we have shown a requirement for SbcCD, the prokaryotic homologue of Rad50/Mre11. This is the first demonstration that, like Rad50/Mre11, SbcCD is required for recombination in a wild-type cell. Our work suggests that the SbcCD-Rad50/Mre11 family of proteins, which have two globular domains separated by a long coiled-coil linker, is specifically required for the co-ordination of double-strand break repair reactions in which two DNA ends are required to recombine at one target site.     

Induction of chromosomal damage by restriction endonuclease in CHO cells porated with streptolysin O.

A procedure is described for the poration of living CHO cells with the bacterial cytotoxin streptolysin O (SLO) which allows the introduction into cells of the restriction endonuclease Pvu II to mimic and model the effects of ionising radiation in causing chromosomal damage. The dependence of this clastogenic effect of Pvu II on SLO concentration was measured by assaying the formation of micronuclei in cytokinesis-blocked binucleate cells. The optimum concentration was found to be 0.045 U/ml. Using the micronucleus assay, the time-course of expression of chromosome damage was investigated and found to show a biphasic kinetic with time. Using a sampling time of 30 h, a dose-effect curve for micronucleus induction by Pvu II was generated. Using this optimized SLO treatment protocol, the frequency of metaphase chromosome damage was subsequently investigated and found to be also linearly related to Pvu II concentration and total aberrations were approximately double the frequency of micronuclei. The induction and repair kinetics of DNA double-strand breaks were investigated in CHO cells treated with SLO and Pvu II using the neutral filter elution technique at pH 9.6. The data presented show that SLO can be used as an alternative method for porating cells to allow the introduction of restriction endonucleases into cells.     

Repair of chromosomal double-strand breaks by precise ligation in human cells

Double-strand breaks (DSBs), a common type of DNA lesion, occur daily in human cells as a result of both endogenous and exogenous damaging agents. DSBs are repaired in two general ways: by the homology-dependent, error-free pathways of homologous recombination (HR) and by the homology-independent, error-prone pathways of nonhomologous end-joining (NHEJ), with NHEJ predominating in most cells. DSBs with compatible ends can be re-joined in vitro with DNA ligase alone, which raises the question of whether such DSBs require the more elaborate machinery of NHEJ to be repaired in cells. Here we report that chromosomal DSBs with compatible ends introduced by the rare-cutting endonuclease, ISceI, are repaired by precise ligation nearly 100% of the time in human cells. Precise ligation depends on the classical NHEJ components Ku70, XRCC4, and DNA ligase IV, since siRNA knockdowns of these factors significantly reduced the efficiency of precise ligation. Interestingly, knockdown of the tumor suppressors p53 or BRCA1 showed similar effects as the knockdowns of NHEJ factors. In contrast, knockdown of components involved in alternative NHEJ, mismatch repair, nucleotide excision repair, and single-strand break repair did not reduce precise ligation. In summary, our results demonstrate that DSBs in human cells are efficiently repaired by precise ligation, which requires classical NHEJ components and is enhanced by p53 and BRCA1.     

Induction of chromosomal aberrations in the CHO mutant EM9 and its parental line AA8 by EcoRI restriction endonuclease: electroporation experiments

EcoRI restriction endonuclease (RE), which produces cohesive-ended double-strand breaks (dsb) in DNA, was tested in the ethyl methanesulfonate- and X-ray-sensitive CHO mutant EM9 and its parental cell strain AA8 for its chromosomal aberration-inducing effect. The RE was efficiently introduced by electroporation into AA8 cells, while the mutant cells showed a very high sensitivity to electroporation, which consistently resulted in cell death. Nevertheless, the incubation of EM9 cells in the presence of EcoRI, without electroporation, was sufficient to induce about three times the chromosome damage observed in the electroporated parental cell line AA8 for any given dose of the RE.     

Chromosomal aberrations induced by the restriction endonucleases EcoR I, Pst I, Sal I and Bam HI in CHO cells

4 widely used cohesive end-producing restriction endonucleases (REs), EcoR I, Pst I, Sal I and Bam HI were tested in CHO cells for their aberration-inducing effects. It was demonstrated that all these REs significantly increased the frequencies of aberrant cells, the aberration frequencies per cell and the aberration frequencies per chromosome. The effects of REs on chromosomal aberrations are similar to ionizing radiation, but more minutes and interchange figures are observed. Polyploid cells are more susceptible to RE treatment, an interesting finding which may be explained by the mechanisms leading to the formation of polyploid cells.     

Influence of bromodeoxyuridine substitution of thymidine on sister-chromatid exchanges and chromosomal aberrations induced by restriction endonucleases

Different levels of replacement of thymidine by 5-bromodeoxyuridine in mammalian DNA have been used to analyze restriction endonuclease-dependent induction of sister-chromatid exchanges and chromosomal aberrations. Data regarding enzyme action in whole cells and in isolated nuclei are presented and discussed. The results indicate a lack of correlation between enzyme effectiveness and the degree of 5-bromodeoxyuridine substitution in the target sequences, specific to the tested restriction endonucleases.     

High chromosomal sensitivity of Chinese hamster xrs 5 cells to restriction endonuclease induced DNA double-strand breaks

The cytogenetic effects of restriction endonucleases (RE) and X-rays were examined in the radiosensitive mutant Chinese hamster cell line xrs 5 and its normal parental line CHO K1. Cells were permeabilized with Sendai virus and exposed to Pvu II and Eco RV which induce blunt-ended double-strand breaks (dsb) in the DNA of cells, or Bam H1 and Eco R1 which induce cohesive-ended dsb with a four-base overlap. Treated cells were then assayed for the presence of metaphase chromosomal aberrations by sampling at multiple fixation times and in experiments where cells were exposed to graded series of RE concentrations. Exposure to X-rays or RE causing blunt-ended dsb was found to be between two and three times more effective in xrs 5 than in CHO K1 cells. We interpret this higher chromosomal sensitivity of xrs 5 cells as reflecting the reported defect in dsb repair in xrs 5. Both xrs 5 and CHO K1 cells yielded less aberrations after exposure to Bam H1 or Eco R1 than after exposure to Pvu II or Eco RV, confirming our previous results and demonstrating that cohesive-ended dsb are less damaging than blunt-ended dsb. Multiple fixation time experiments showed that the higher sensitivity of xrs 5 was evident at several different sampling times after treatment. Similarly the low yield of aberrations after exposure of cells to Bam H1 was evident at all sampling times. Overdispersion of chromosomal aberrations was observed in samples exposed to RE. This is thought to be due to a non-uniform permeabilization of the cell population to RE. Our results indicate that RE-induced dsb are handled by cells in a similar way to those arising during X-ray exposure.     

Cyclin A overexpression induces chromosomal double-strand breaks in mammalian cells

Cyclin A is a major regulator in vertebrate cell cycle, associated with cyclin-dependent kinase (Cdk), and involved in S-phase progression and entry into mitosis. It has been known that cyclin A overexpression not only causes premature S-phase entry but also induces prolongation of S phase. Here we show that ectopic expression of cyclin A leads to extensive γ?H2AX focus formation, which is indicative of DNA double-strand breaks. Likewise, cyclin E, but not cyclin B1 and cyclin D1, also induced the γ?H2AX focus formation, suggesting that these DNA lesions may be induced via aberrant DNA replication process. Moreover, the γ?H2AX focus formation was suppressed by co-expressing p21Cip1/Waf1 or dominant-negative Cdk2 mutant, suggesting that aberrant cyclin A-Cdk2 activation induces the chromosomal double-strand breaks.     

Induction of sister-chromatid exchanges by restriction endonucleases

Restriction endonucleases Cfo 1, Pvu II, Sma I, Hpa II, Taq I and Hae III were tested for their ability to induce SCEs in CHO cells. The results indicate that the DNA double-strand breaks induced during S-phase by these enzymes lead to an increase in the frequencies of SCEs.     

Molecular recombination and the repair of DNA double-strand breaks in CHO cells.

Molecular recombination and the repair of DNA double-strand breaks (DSB) have been examined in the G-0 and S phase of the cell cycle using a temperature-sensitive CHO cell line to test i) if there are cell cycle restrictions on the repair of DSB's' ii) the extent to which molecular recombination can be induced between either sister chromatids or homologous chromosomes and iii) whether repair of DSB's involves recombination (3). Mitomycin C (1-2 micrograms/ml) or ionizing radiation (50 krad) followed by incubation resulted in molecular recombination (hybrid DNA) in S phase cells. Approximately 0.03 to 0.10% of the molecules (number average molecular weight: 5.6 x 10(6) Daltons after shearing) had hybrid regions for more than 75% of their length. However, no recombination was detected in G-0 cells. Since the repair of DSB was observed in both stages with more than 50% of the breaks repaired in 5 hours, it appears that DSB repair in G-0 cells does not involve recombination between homologous chromosomes. The possibility is not excluded that repair in G-0 cells involves only small regions (less than 4 x 10(6) Daltons).     

Induction and rejoining of DNA double-strand breaks in bladder tumor cells

The induction and rejoining of radiation-induced double-strand breaks (DSBs) in cells of six bladder tumor cell lines (T24, UM-UC-3, TCC-SUP, RT112, J82, HT1376) were measured using the neutral comet assay. Radiation dose-response curves (0-60 Gy) showed damage (measured as mean tail moment) for five of the cell lines in the same rank order as cell survival (measured over 0-10 Gy), with the least damage in the most radioresistant cell line. Damage induction correlated well with clonogenic survival at high doses (SF10) for all six cell lines. At the clinically relevant dose of 2 Gy, correlation was good for four cell lines but poor for two (TCC-SUP and T24). The rejoining process had a fast and slow component for all cell lines. The rate of these two components of DNA repair did not correlate with cell survival. However, the time taken to reduce the amount of DNA damage to preirradiated control levels correlated positively with cell survival at 10 Gy but not 2 Gy; radioresistant cells rejoined the induced DSBs to preirradiation control levels more quickly than the radiosensitive cells. Although the results show good correlation between SF10 and DSBs for all six cell lines, the lack of correlation with SF2 for TCC-SUP and T24 cells would suggest that a predictive test should be carried out at the clinically relevant dose. At present the neutral comet assay cannot achieve this.     

Generation of highly site-specific DNA double-strand breaks in human cells by the homing endonucleases I-PpoI and I-CreI

We have determined the ability of two well-characterized eukaryotic homing endonucleases, I-PpoI from the myxomycete Physarum polycephalum and I-CreI from the green alga Chlamydomonas reinhardtii, to generate site-specific DNA double-strand breaks in human cells. These 18-kDa proteins cleave highly conserved 15- or 24-bp rDNA homing sites in their respective hosts to generate homogeneous 4-base, 3' ends that initiate target intron transposition or "homing." We show that both endonucleases can be expressed in human cells and can generate site-specific DNA double-strand breaks in 28S rDNA and homing site plasmids. These endonuclease-induced breaks can be repaired in vivo, although break repair is mutagenic with the frequent generation of short deletions or insertions. I-PpoI and I-CreI should be useful for analyzing DNA double-strand break repair in human cells and rDNA.     

Increased repair of gamma-induced DNA double-strand breaks at lower dose-rate in CHO cells

DNA double-strand breaks (DSBs) are highly cell damaging. We asked whether for a given dose a longer irradiation time would be advantageous for the repair of DSBs. Varying the gamma-irradiation dose and its delivery time (0.05 Gy/min low dose-rate (LDR) compared with 3.5 Gy/min high dose-rate), confluent Chinese hamster ovary cells (CHO-K1) and Ku80 mutant cells (xrs-6) deficient in nonhomologous end-joining (NHEJ) were irradiated in agarose plugs at room temperature using a cesium-137 gamma-ray source. We used pulsed-field gel electrophoresis (PFGE) to measure DSBs in terms of the fraction of activity released (FAR). At LDR, one third of DSBs were repaired in CHO-K1 but not in xrs-6 cells, indicating the involvement of NHEJ in the repair of gamma-induced DSBs at a prolonged irradiation incubation time. To improve DSB measurements, we introduced in our PFGE protocol an antioxidant at the cell lysis step, thus avoiding free-radical side reactions on DNA and spurious DSBs. Addition of the metal chelator deferoxamine (DFO) decreased more efficiently the basal DSB level than did reduced glutathione (GSH), showing that measuring DSBs in the absence of DFO reduces precision and underestimates the role of NHEJ in the dose-rate effect on DSB yield.